scholarly journals Glycan analysis of human neutrophil granules implicates a maturation-dependent glycosylation machinery

2020 ◽  
Author(s):  
Vignesh Venkatakrishnan ◽  
Regis Dieckmann ◽  
Ian Loke ◽  
Harry Tjondro ◽  
Sayantani Chatterjee ◽  
...  
Keyword(s):  
Gradient Centrifugation ◽  
Effector Proteins ◽  
Protein Glycosylation ◽  
Sialyl Lewis X ◽  
Human Neutrophils ◽  
Immune Functions ◽  
Lewis X ◽  
Percoll Density Gradient Centrifugation ◽  
Percoll Density Gradient ◽  
And Function

AbstractProtein glycosylation is essential to trafficking and immune functions of human neutrophils. During granulopoeisis in the bone marrow, distinct neutrophil granules are successively formed. Distinct receptors and effector proteins, many of which are glycosylated, are targeted to each type of granule according to their time of expression, a process called ‘targeting-by-timing’. Therefore, these granules are time capsules reflecting different times of maturation that can be used to understand how glycosylation evolves during granulopoiesis. Herein, neutrophil subcellular granules were fractionated by Percoll density gradient centrifugation and N- and O-glycans present in each compartment were analyzed by liquid chromatography and tandem mass spectrometry. We found abundant paucimannosidic N-glycans and lack of O-glycans in early-formed azurophil granules (AG), whereas later-formed specific and gelatinase granules (SG and GG) contained complex N- and O-glycans with remarkably elongated N-acetyllactosamine repeats with Lewis-x and sialyl-Lewis-x epitopes. Many glycans identified are unique to neutrophils and their complexity increased progressively from AG to SG and then to GG, suggesting temporal changes in the glycosylation machinery indicative of ‘glycosylation-by-timing’ during granulopoiesis. In summary, this comprehensive neutrophil granule glycome map, the first of its kind, highlights novel granule-specific glycosylation features and is a crucial first step towards a better understanding of the mechanisms regulating protein glycosylation during neutrophil granulopoiesis and a more detailed understanding of neutrophil biology and function.

scholarly journals Glycan analysis of human neutrophil granules implicates a maturation-dependent glycosylation machinery

2020 ◽  
Vol 295 (36) ◽  
pp. 12648-12660 ◽  
Author(s):  
Vignesh Venkatakrishnan ◽  
Régis Dieckmann ◽  
Ian Loke ◽  
Harry C. Tjondro ◽  
Sayantani Chatterjee ◽  
...  
Keyword(s):  
Gradient Centrifugation ◽  
Effector Proteins ◽  
Protein Glycosylation ◽  
Sialyl Lewis X ◽  
Human Neutrophils ◽  
Immune Functions ◽  
Lewis X ◽  
Percoll Density Gradient Centrifugation ◽  
Percoll Density Gradient ◽  
And Function

Protein glycosylation is essential to trafficking and immune functions of human neutrophils. During granulopoiesis in the bone marrow, distinct neutrophil granules are successively formed. Distinct receptors and effector proteins, many of which are glycosylated, are targeted to each type of granule according to their time of expression, a process called “targeting by timing.” Therefore, these granules are time capsules reflecting different times of maturation that can be used to understand the glycosylation process during granulopoiesis. Herein, neutrophil subcellular granules were fractionated by Percoll density gradient centrifugation, and N- and O-glycans present in each compartment were analyzed by LC–MS. We found abundant paucimannosidic N-glycans and lack of O-glycans in the early-formed azurophil granules, whereas the later-formed specific and gelatinase granules and secretory vesicles contained complex N- and O-glycans with remarkably elongated N-acetyllactosamine repeats with Lewis epitopes. Immunoblotting and histochemical analysis confirmed the expression of Lewis X and sialyl-Lewis X in the intracellular granules and on the cell surface, respectively. Many glycans identified are unique to neutrophils, and their complexity increased progressively from azurophil granules to specific granules and then to gelatinase granules, suggesting temporal changes in the glycosylation machinery indicative of “glycosylation by timing” during granulopoiesis. In summary, this comprehensive neutrophil granule glycome map, the first of its kind, highlights novel granule-specific glycosylation features and is a crucial first step toward a better understanding of the mechanisms regulating protein glycosylation during neutrophil granulopoiesis and a more detailed understanding of neutrophil biology and function.

Purification of dispersed rat adrenal zona glomerulosa cells by Percoll density gradient centrifugation and the isolation of a population of cells highly responsive to adrenocorticotrophin

1986 ◽  
Vol 109 (3) ◽  
pp. 351-NP ◽  
Author(s):  
F. W. Chu ◽  
P. J. Hyatt
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Zona Glomerulosa ◽  
Zona Fasciculata ◽  
Sephadex Column ◽  
Percoll Density Gradient Centrifugation ◽  
Percoll Density Gradient ◽  
Glomerulosa Cells ◽  
Unit Gravity Sedimentation

ABSTRACT Percoll density gradient centrifugation is a simple, inexpensive and convenient method to eliminate contaminating zona fasciculata (ZF) cells from unpurified rat adrenal capsular glomerulosa (ZG) cell preparations (with less than 0·1% ZF cells in the final cell preparation). Basal steroid (aldosterone and corticosterone) output by the purified (PG) cells was unchanged. These purified cells, although free from ZF contamination, were more highly responsive than expected to ACTH (3 nmol/l). When PG cells were further separated by Sephadex column filtration, the filtered PG cells exhibited the steroidogenic response of ZG cells purified by unit gravity sedimentation and Sephadex column filtration, i.e. reduced basal steroid output and an ACTH response reduced to that stimulated by K+ (8·4 mmol/l). Although the cells retained in the column resembled the filtered PG cells ultrastructurally, they showed unchanged basal steroid output and a high ACTH response with increased latepathway activity (the conversion of corticosterone to aldosterone). By combining Percoll density gradient centrifugation and Sephadex column filtration we have a method for the isolation and study of both the high-and low-response rat ZG cells which are free from ZF contamination. J. Endocr. (1986) 109, 351–358

Oxidative stress in isolated rat renal proximal and distal tubular cells

1990 ◽  
Vol 259 (2) ◽  
pp. F338-F347 ◽  
Author(s):  
L. H. Lash ◽  
J. J. Tokarz
Keyword(s):  
Rat Kidney ◽  
Gradient Centrifugation ◽  
Buthionine Sulfoximine ◽  
Oxidative Injury ◽  
Cell Types ◽  
Protective Role ◽  
Cortical Cells ◽  
Percoll Density Gradient Centrifugation ◽  
Tert Butyl Hydroperoxide ◽  
Percoll Density Gradient

Suspensions of purified proximal tubular (PT) and distal tubular (DT) cells were isolated from rat kidney cortical cells by Percoll density-gradient centrifugation and were used to investigate susceptibility of these regions of the nephron to oxidative injury. Exposure to tert-butyl hydroperoxide (tBH), menadione (MD), or H2O2 produced significantly greater cytotoxicity, as assessed by leakage of lactate dehydrogenase, in DT cells than in PT cells. The order of cytotoxic potency in both cell types was MD greater than tBH greater than H2O2. Preincubation of PT and DT cells with 5 mM glutathione (GSH) or 5 mM dithiothreitol delayed tBH-induced cytotoxicity, indicating a protective role of GSH. Addition of buthionine sulfoximine and acivicin with GSH, to inhibit GSH synthesis and degradation, eliminated the protective effect of GSH, indicating that protection by GSH in DT cells is not dependent on uptake of the intact tripeptide. Incubation of both PT and DT cells with tBH resulted in oxidation of GSH to glutathione disulfide. Activities of five detoxication enzymes were significantly higher in PT cells, indicating that a diminished ability to detoxify reactive metabolites may contribute to the higher intrinsic susceptibility of DT cells to oxidative injury.

scholarly journals Visualizing the Integrity of Chloroplast Envelope by Rhodamine and Nile Red Staining

2021 ◽  
Vol 12 ◽  
Author(s):  
Jinjie An ◽  
Xin Miao ◽  
Lulu Wang ◽  
Xu Li ◽  
Xiaomin Liu ◽  
...  
Keyword(s):  
Fluorescent Dyes ◽  
Gradient Centrifugation ◽  
Nile Red ◽  
Middle Layer ◽  
Chloroplast Envelope ◽  
The Past ◽  
Biochemical Methods ◽  
Percoll Density Gradient Centrifugation ◽  
Percoll Density Gradient ◽  
Isolated Chloroplasts

Chloroplasts are essential organelles in plant cells with many important functions. Chloroplasts isolated by Percoll density gradient centrifugation are widely used in the study of chloroplasts. The intactness of isolated chloroplasts is necessary for many of the experiments. In the past, those isolated chloroplasts were either simply believed to be intact or had to be analyzed by indirect biochemical methods. Here we show a new method to check the intactness of isolated chloroplasts by staining their envelope with fluorescent dyes, Rhodamine or Nile red, and then observing them with a fluorescence microscope. With this method, broken chloroplasts and intact chloroplasts can be distinguished easily and their integrity can be checked in a few minutes. Results of this method agreed well with those of biochemical methods. Moreover, we have also found that sometimes the middle layer chloroplasts from the Percoll gradient centrifugation could be mostly broken, which could cause mistakes in the experiment. With our method, this problem can be easily found. This chloroplast envelope staining method can be used in the preparation of isolated chloroplasts to ensure the intactness.

scholarly journals Subpopulations of rat hepatocytes separated by Percoll density-gradient centrifugation show characteristics consistent with different acinar locations

1994 ◽  
Vol 304 (2) ◽  
pp. 617-624 ◽  
Author(s):  
J C Osypiw ◽  
R L Allen ◽  
D Billington
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Rat Hepatocytes ◽  
Marker Enzymes ◽  
Cytotoxic Effects ◽  
Percoll Density Gradient Centrifugation ◽  
Cell Layers ◽  
Density Band ◽  
Percoll Density Gradient

Freshly isolated viable rat hepatocytes were separated into five subpopulations on shallow discontinuous Percoll density gradients. The periportal marker enzymes alanine aminotransferase (ALT), malate dehydrogenase (MDH) and lactate dehydrogenase (LDH) showed gradients of increasing activity from the subpopulation of least density (band 1, rho = 1.07 g/ml) to the subpopulation of greatest density (band 5, rho = 1.09 g/ml). The perivenous marker enzymes pyruvate kinase (PK) and glutamate dehydrogenase (GDH) showed gradients of decreasing activity from band-1 cells to band-5 cells. Glutamine synthetase (GS), which is confined to the two or three cell layers around the hepatic venule, was almost entirely restricted to band-1 hepatocytes. Band-5: band-1 ratios of enzyme activity were as follows: ALT, 8.0; LDH, 2.1; MDH, 1.6; GDH, 0.7; PK, 0.2; GS, 0.01. Band-5:band-1 ratios for ALT, LDH, PK and GS were maintained after culture of subpopulations in identical conditions for up to 72 h, whereas the ratios for MDH and GDH decreased and increased respectively towards unity. Band-1 hepatocytes exhibited greater cytotoxicity than band-5 cells after incubation with carbon tetrachloride or paracetamol. These perivenous-selective toxins produced greater decreases in cell viability and greater release of ALT and LDH from band-1 hepatocytes than from band-5 hepatocytes. Conversely, band-5 hepatocytes were more susceptible than band-1 hepatocytes to the cytotoxic effects of 1-naphthylisothiocyanate and methotrexate (known periportal-selective toxins). It is concluded that band-5 hepatocytes are enriched in periportal cells, whereas band-1 hepatocytes are enriched in perivenous cells. Isolation of hepatocyte subpopulations by Percoll density-gradient centrifugation has the considerable advantage that periportal and perivenous cells can be obtained from the same liver.

H(+)-ATPases of renal cortical and medullary endosomes are differentially sensitive to Sch-28080 and omeprazole

1994 ◽  
Vol 266 (6) ◽  
pp. F868-F877 ◽  
Author(s):  
I. Sabolic ◽  
D. Brown ◽  
J. M. Verbavatz ◽  
J. Kleinman
Keyword(s):  
Gradient Centrifugation ◽  
Kidney Tissue ◽  
Renal Medulla ◽  
Fluorescein Isothiocyanate ◽  
Similar Degree ◽  
Percoll Density Gradient Centrifugation ◽  
Fluorescence Quench ◽  
Dependent Inhibition ◽  
Tissue Homogenates ◽  
Percoll Density Gradient

Adenosinetriphosphatase (ATPase) activity stimulated by K+ and inhibited by Sch-28080 (SCH), omeprazole (OME), and vanadate has been measured in microsomes from mammalian renal medulla and attributed to a kidney isoform of the H(+)-K(+)-ATPase. To determine whether the H(+)-K(+)-ATPase inhibitors could also inhibit the vacuolar (V)-type H(+)-adenosinetriphosphatase (H(+)-ATPase, i.e., H+ pump) in mammalian intracellular vesicles, we examined their effects on bafilomycin-sensitive acidification in renal cortical vesicles (CEV) and medullary endocytic vesicles (MEV). Rats were injected with fluorescein isothiocyanate-labeled dextran, and labeled endosomes were enriched from kidney tissue homogenates by differential and Percoll density gradient centrifugation. In the CEV, the V-type H+ pump was inhibited 25% by SCH and 30% by OME (100 microM each). Whereas the inhibition by OME was concentration and time dependent, the inhibition by SCH was only concentration dependent. Inhibition by these compounds was similar in the presence of 50 mM K+ (in = out) and in the complete absence of K+, thus ruling out a significant involvement of H(+)-K(+)-ATPase-mediated acidification. Inhibition, however, was not observed with 10 microM SCH and OME. The sensitivity of the V-type H+ pump to 100 microM SCH and OME in CEV was confirmed by the comparable inhibitions of intravesicular acidification observed in acridine orange fluorescence quench studies and by inhibition of Pi liberation in an ATPase assay. We also found that the V-type H+ pump in isolated rat liver endosomes is sensitive to 100 microM SCH and OME to a similar degree. In the MEV, acidification was only weakly affected by 100 microM SCH and OME, thus suggesting that H(+)-ATPases in endosomes from cortical and medullary tubules are different, possibly due to a previously described selective expression of subunit isoforms. Our finding indicates the importance of using low concentrations (< 10 microM) of OME and SCH in studies of H(+)-K(+)-ATPase in nongastric tissues to avoid misinterpretation of the data due to nonspecific inhibition of V-type H(+)-ATPases.

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